There are a variety of different applications in which a sample-to-sequence result in a short period of time would have great utility. These applications may include rapid, near point-of-care or laboratory, identification of mutations that confer antibiotic resistance, sequencing of viral genes, identification of tissue for forensic purposes, tissue typing for organ transplantation, and identification of alleles related to rates of drug metabolism, for example.
For example, for bacterial pathogens implicated in sepsis, it is useful to know whether the bacteria carry extended spectrum beta-lactamases (ESBLs) that make them resistant to extended spectrum cephalosporins. These ESBLs result from mutations in the TEM-1, TEM-2, SHV, and other beta lactamase genes. There are numerous other genes that can confer an antibiotic resistance phenotype, whose expression or activity is modulated by point mutations in the coding or regulatory regions of these genes. While single known point mutations are easy to assay using standard PCR techniques, mutations in genes that have multiple alleles may be more difficult to identify.
Likewise there are numerous instances in which the sequence of a viral gene can indicate both the presence of that virus in a human sample and whether that virus is resistant to antiviral compounds, and thus indicating what treatment should be started, continued, or stopped. This is true for Hepatitis C, Hepatitis B, and HIV. While time-to-result is not quite as pressing for these chronic viral infections, time is of the essence for other viral infections, such as influenza. With influenza and other acute viral infections, there are an increasing number of well-characterized point mutations that confer resistance to the FDA approved neuraminidase inhibitors. Ideally, treatment with a neuraminidase inhibitor should start as soon as possible after the virus is detected, but it is important that the correct inhibitor be used.
In addition to pathogen detection and identification, there are several instances in which determining the sequence of a human gene or genes quickly is important. These include identification of human tissue for forensic purposes, illustratively by mapping the length of Short Tandem Repeats (STRs). This identification is currently performed by sizing PCR amplicons, but it can also be done by sequencing. A quick sample-to-sequence method would be helpful in many such cases.
Another example is HLA typing an organ for transplantation. Organs to be donated may come from a recently deceased individual. It is important to get the organ delivered quickly to the transplant recipient that has the best MHC match to the donor. The most accurate way to do this is to sequence the MHC genes that govern transplant rejection and then match these sequences to a national registry of recipient MHC types. A quick sample-to-sequence method would be helpful in many organ donation cases.
Yet another example is identification of cytochrome P450 alleles that determine rates of drug metabolism. It is known that cytochrome P450 enzymes in the liver metabolize foreign compounds for excretion. Multiple proteins in this family are known, and different alleles of each are present in different populations and individuals. For example, some people carry alleles for enzymes that metabolize warfarin very fast and some metabolize warfarin much more slowly. Knowing a patient's genotype permits a doctor to decide how much warfarin or other such drugs to administer to achieve a certain level of drug in the blood stream.
Other non-limiting examples where sequencing could be useful includes cancer genes, as well as other infectious agents such as tuberculosis. Many other examples are possible, as are uses both in clinical diagnostics and in other fields.
Polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA generated (the amplicon) is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR, it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR employs a thermostable polymerase, dNTPs (deoxynucleotide triphosphates), and a pair of primers (oligonucleotides). Existing PCR techniques and other amplification methods may be combined with next generation sequencing (NGS) to provide quick sample-to-sequence results.
Traditional nucleic acid sequencing techniques employ either chemical cleavage at a specific base (Maxim-Gilbert method) or chain termination using dideoxynucleotides (Sanger sequencing). Next-generation sequencing involves high-throughput sequencing technologies some of which parallelize the sequencing process, producing thousands to millions of sequences concurrently, often detecting as each nucleotide is added to each individual strand. Various next-generation systems are currently available.